Fig. 4: Impact of LAMA-mediated depalmitoylation on cellular distribution of GFP-Spry2 in HEK293T cells.

A Impact of trimethoprim (TMP, 10 µM, applied overnight) on APT2-LAMA-G97 mediated depalmitoylation of eGFP-Spry2, assessed using acyl-RAC. Data are means ± SEM from n = 7 independent experiments. Statistical comparison: unpaired two-tailed t-test. B Subcellular distribution of eGFP-Spry2 in the absence of nanobody (left), the presence of nanobody (middle), and the presence of nanobody and trimethoprim (10 µM, applied overnight). Scale bar: 10 µm. Experiment was repeated independently five times with similar results. C Impact of trimethoprim withdrawal on eGFP-Spry2 cellular distribution assessed by fractionation. Cyt: cytoplasmic fraction, WCL: whole cell lysate, -1hr: trimethoprim (10 µM, applied overnight) then withdrawn for 1 h, o/n: trimethoprim (10 µM) applied overnight. Data are means ± SEM from n = 8 independent experiments. Statistical comparisons: one-way ANOVA followed by Dunnett’s multiple comparisons test. D Live cell imaging to visualise eGFP-Spry2 redistribution upon trimethoprim withdrawal. Snapshots taken at 5 min intervals following withdrawal of trimethoprim from cells expressing eGFP-Spry2 and APT2-LAMA-G97. The number if the upper left corner of each image indicates the time (minutes) since TMP withdrawal. Scale bar: 10 µm. Experiment was repeated independently five times with similar results. E Palmitoyl proteome analysis of Flp-In T-REx cells stably expressing tetracycline-inducible APT2-G97-LAMA. Data are presented as log2 fold change values with a cut-off of 0.58 and a q-value threshold of 0.05. Statistical comparisons: moderated, two-sided t-tests with adjustments for multiple comparisons using the Benjamini-Hochberg procedure. Palmitoylation of 25 proteins (5% of the palmitoyl proteome) is reduced and palmitoylation of 2 proteins is increased following induction of the nanobody chimera expression. Data from n = 3 independent experiments. Source data are provided as a Source Data file.